The photoreceptor synaptic triad, consisting of a photoreceptor terminal, bipolar cell dendrites and horizontal cell endings, is a specialized synaptic complex of great importance. Physiologically, this is the site of initial transfer of visual information from photoreceptors and the fidelity of information transfer is critically important for visual processing. Horizontal cells mediate inhibitory feedback in the outer retina at bipolar cell dendrites and photoreceptor terminals;however, the mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. For instance, horizontal cell endings in the synaptic triad have relatively few small, clear-core vesicles and they lack conventionally defined presynaptic membrane specializations. In contrast, these cells express established synaptic vesicle proteins, including the vesicular GABA transporter (VGAT) and SV2A, a complement of synaptic proteins, including SNAP-25 and complexin, that are associated with exocytosis, and L-type Ca2+ channels, suggesting GABA is released by a vesicular mechanism. This mechanism differs from a previously established GABA plasmalemmal transporter mechanism described for non-mammalian horizontal cells. The long-term objective of this project is to understand the functional role of mammalian horizontal cells in visual information processing. This application will address this objective by examining the hypothesis that the cellular structure and biochemical machinery that mediate vesicular transmitter release are present in mammalian horizontal cells. This hypothesis will be examined as follows: Specific aim 1: Characterize vesicles in horizontal cell endings in the synaptic triad. Vesicle type, number and distribution in horizontal cell dendrites that innervate cone pedicles and axonal terminals that innervate rod spherules will be examined with conventional electron microscopy. Specific aim 2: Examine the distribution of the principal vesicular proteins which are involved in regulated exocytosis in horizontal cell endings by determining the expression of a) VGAT and b) VAMP isoforms, SV2A, synaptotagmin 1 and 2, Rab3 isoforms, and the vacuolar proton pump with RT-PCR and immunohistochemistry. Specific aim 3: Examine the distribution of key synaptic proteins, which participate in vesicle docking, priming and fusion in regulated transmitter release, in horizontal cell endings by determining the expression of syntaxin, SNAP-25 and complexin 1-4, Munc 13-1 and 18, and RIM1 with RT-PCR and immunohistochemistry. These studies will further elucidate the structural and biochemical basis of transmitter release from horizontal cells, thus providing a better understanding of the functional role of horizontal cells in the outer retina. These findings will aid in the development of therapeutic strategies for the treatment of pathological changes in the outer retina related to retinal disease, such as macular degeneration.